Use of a ribozyme to join nucleic acids and peptides

ABSTRACT

Engineered mRNA useful in producing libraries of engineered mRNAs, polypeptide-engineered mRNA conjugates and diverse encoded polypeptide libraries, as well as novel ribozymes that join an mRNA to the translation product of the mRNA and methods of identifying members of diverse encoded polypeptide libraries which exhibit a desired activity. Also described are polypeptide-nucleic acid tag conjugates, methods of producing the conjugates and uses therefor.

FUNDING

The subject invention was supported in part by Postdoctoral FellowshipGrant number DBJ-9750048 from the National Science Foundation and theAlfred P. Sloan Foundation.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application60/082,256, entitled A New Method for Generating Diverse Libraries ofEncoded Polypeptides, by Donald Scott Baskerville and David P. Bartel,filed Apr. 17, 1998. The entire teachings of U.S. 60/082,256 areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Large combinatorial libraries of biopolymers are starting points forisolating new enzymes, binding motifs and other useful molecules. Forexample, current technologies can generate populations of nucleic acidswith complexities on the order of 10¹⁵ molecules and then isolate andidentify a single molecule with a desired activity. Random polypeptidepopulations have greater chemical diversity than do polynucleotides,making them an attractive alternative to nucleic acids. Current systemsare limited in their ability to easily generate large complex librariesof polypeptides that are in a form that allows the isolation andidentification of rare molecules with a desired activity.

SUMMARY OF THE INVENTION

Described herein are ribozymes which join a nucleic acid (RNA or DNA) toa polypeptide, the resulting products and uses for these products. Inone embodiment of the invention, a ribozyme which joins RNA to apolypeptide is used to produce a diverse library (or collection) ofencoded polypeptides, in which library members arepolypeptide-engineered mRNA conjugates. The polypeptide-engineered mRNAconjugates comprise an engineered mRNA, which is described below, andthe translation product of the engineered mRNA. Diverse libraries ofencoded polypeptides of the present invention can be screened toidentify and isolate library members which are conjugates in which thepolypeptide exhibits desired characteristics or properties (e.g.,binding to a molecule or compound of interest, enzymatic activity). In asecond embodiment, a ribozyme which joins RNA to a polypeptide is usedto produce polypeptides, each of which is linked to and, thus, taggedby, a specific nucleic acid. The resulting nucleic acid-taggedpolypeptides comprise a polypeptide (which is to be tagged by a nucleicacid); a peptide substrate of the ribozyme used to join the nucleic acidto the polypeptide; ribozyme RNA and a nucleic acid tag. The presence ofthe nucleic acid tag is useful, for example, in detecting, isolating,separating, identifying or purifying the polypeptide to which it islinked and in changing the properties (e.g., solubility) of thepolypeptides. An unlimited number of nucleic acid tags can be made,which overcomes a limitation of presently-available methods, which relyon a small repertoire of affinity tags, as well as sometimesincompatible incubation protocols for each tag. Such ribozymes, methodsof using them, the resulting products and methods of identifying,detecting, isolating, separating, purifying and/or using these productsare described in detail herein.

One embodiment of the present invention relates to the use of ribozymesdescribed herein to produce a diverse library or collection of encodedpolypeptides in which the members are polypeptide-engineered mRNAconjugates; engineered mRNA; methods of producing diverse libraries ofencoded polypeptides; methods of identifying target members of thelibraries which are polypeptide-engineered mRNA conjugates which exhibita desired activity or characteristic; target members and the components(polypeptide fragment and engineered mRNA fragment) of target members;and isolated ribozymes which join an mRNA to the translation product ofthe mRNA.

Polypeptide-engineered mRNA conjugates of the present invention comprisean engineered mRNA and the translation product of the engineered mRNA.Engineered mRNA of the present invention is one component of theconjugates which make up the diverse libraries of encoded polypeptidesand is used to produce the conjugates. Members of the diverse libraryare polypeptide-engineered mRNA conjugates produced by in vitrotranslation of engineered mRNA. The engineered mRNA comprises: (a) aribozyme RNA which specifically covalently links to a peptide; (b) acoding region for the peptide, referred to as a peptide tag, with whichthe ribozyme RNA specifically covalently links; (c) a coding region fora diverse polypeptide; and (d) two PCR primer-binding sites. In oneembodiment, the engineered mRNA comprises, in order from the 5' to the3' end, a ribozyme segment which comprises a first PCR primerbinding-site and a motif that interacts specifically with a peptide; acoding region for the peptide with which the motif interacts; a codingregion for a diverse polypeptide and a second PCR primer-binding site.

The ribozyme RNA which specifically covalently links to a peptide ispresent in (is a component or segment of) a ribozyme which is either acontiguous sequence (the entire ribozyme is a contiguous sequence) orcomprised of two noncontiguous components: one which comprises theribozyme RNA which specifically covalently links to the peptide and onewhich comprises the remainder of the ribozyme sequence. In the lattercase, the ribozyme RNA which covalently links to a peptide is ofsufficient length and appropriate composition to covalently link to thepeptide tag in the presence of the remainder of the ribozyme sequence,under the conditions used to produce the diverse libraries. Theremaining ribozyme sequence is the ribozyme sequence which, incombination with the component which covalently links to the peptidetag, makes up the complete ribozyme. The ribozyme sequence or segmentwhich covalently links to the peptide tag can be as short as onenucleotide in length and can be from any location (e.g., 5' end,internal segment, 3' end) in the ribozyme. In one embodiment, theribozyme segment includes from one to about 18 nucleotides, such as fromthe first to about the 18th nucleotide (from the 5' end) of a ribozyme.In further embodiments, the ribozyme segment is the first 13 to 18nucleotides (from the 5' end) of the ribozyme. (e.g., the first 13, 14,15, 16, 17, or 18 nucleotides from the 5' end). The other component isthe remaining ribozyme sequence (the remainder of the ribozyme which isnecessary to form a functional ribozyme.). The two ribozyme componentsinteract with one another to form a functional (complete) ribozyme underthe conditions used to produce diverse libraries of encodedpolypeptides. The 5' end of the ribozyme RNA optionally comprises threephosphate groups or an mRNA cap, such as a 7-methyl guanosinetriphosphate. Optionally, the engineered mRNA further comprises an RNAlinker between the ribozyme sequence and the tag coding region.Optionally, the engineered mRNA additionally comprises a coding regionfor a peptide linker; this coding region is positioned between thecoding region for the peptide tag and the diverse coding region.Further, the engineered mRNA can include an optional ribosome stallingsite, which is located between the coding region for the diversepolypeptide segment and the second PCR primer-binding site.

In one embodiment, the engineered mRNA comprises (in order from 5' to3'): 1) a ribozyme sequence (also referred to as which comprises a firstPCR primer-binding site and a motif that interacts specifically with apeptide; 2) a coding region for a peptide (referred to as a peptidetag), with which the ribozyme motif interacts specifically; 3) a codingregion for a diverse polypeptide segment and 4) a second PCRprimer-binding site. In addition, in this embodiment, the engineeredmRNA can include one or more of the following optional components: 5'phosphate groups; an RNA linker between the ribozyme sequence and thetag coding region: a coding region for a peptide linker (positionedbetween the tag coding region and the diverse coding region) and aribosome stalling site which is located between the coding region forthe diverse polypeptide segment and the second PCR primer-binding site.

In a specific embodiment, the motif that interacts specifically with apeptide is modified Bovine Immunodeficiency Virus-1 (BIV-1) TAR RNA andthe peptide tag is one with which BIV-1 TAR RNA interacts specificallyby virtue of the BIV-1 Tat sequence embedded within the tag, e.g., theTat tag peptide 1, also referred to as the Tat tag (SEQ ID NO.: 1:MSYSGPRPRGTRGKGRRIRRGGK), or the Tat 2 tag peptide, also referred to asthe Tat 2 tag (SEQ ID NO.: 2: MKYSGPRPRGTRGKGRRIRRGGK). In both the Tattag peptide and the Tat 2 tag peptide, the underlined amino acids are aBIV-1 Tat peptide (SEQ ID NO.: 3). The sequence of the modified BIV-1TAR RNA is: 5' GGA CAG CUC CGA GCA UUC UCG UGU AGC U (SEQ ID NO.: 4).

Also the subject of this invention are isolated ribozymes or ribozymeportions which join an mRNA to a polypeptide, such as the translationproduct of the mRNA. Such ribozymes comprise a motif which specificallycovalently links to a peptide, with the result that the ribozyme isjoined specifically to the peptide. Ribozymes of the present inventioncan be a contiguous sequence or can be comprised of two noncontiguouscomponents which interact with one another to form the completeribozyme, under the conditions used to produce diverse libraries ofencoded polypeptides. The two noncontiguous components are a ribozymesegment which specifically covalently links to a peptide and a ribozymesegment which comprises the remaining ribozyme. As a result, an mRNAwhich comprises such a ribozyme and a coding region for the peptide withwhich the ribozyme interacts will be joined to the translation productof the mRNA (through binding and then covalent linkage of the ribozymeRNA and the peptide). An example of a ribozyme that contains a motifthat interacts specifically with a peptide (modified TAR RNA) and joinsitself specifically to certain peptides (e.g., the Tat tag peptide orthe Tat 2 tag peptide) is specifically described herein.

Members of the encoded polypeptide libraries, referred to as targetmembers, which have desired characteristics (e.g., binding, enzymatic,antigenic characteristics) are also the subject of this invention, asare methods of identifying target members. Polypeptide fragments andribonucleoprotein fragments of members of the libraries, particularlyfragments of target members of the libraries, are further subjects ofthis invention.

The present invention provides a method and reagents for generatingdiverse libraries of encoded polypeptides and for identifying andisolating members of the libraries, particularly members which exhibitrare characteristics and occur in small numbers.

A second embodiment of the present invention relates to the use ofribozymes to join a desired nucleic acid tag to a polypeptide to producea polypeptide tagged with the desired (specific) nucleic acid. Asdescribed above, the ribozyme can comprise a motif which specificallycovalently links to a peptide. The resulting polypeptide-nucleic acidtag conjugate is a subject of the present invention, as are itscomponents, collections or libraries of such conjugates and methods ofmaking and using the conjugates. In this embodiment, a ribozyme whichjoins a polypeptide to a nucleic acid is used as follows to producepolypeptide-nucleic acid tag conjugates: A polypeptide, also referred toas a polypeptide of interest (e.g., a polypeptide to be identified,detected, separated, isolated, purified, altered in characteristic(s)),which is tagged with the peptide substrate of the ribozyme used, isjoined covalently, as described below, to the nucleic acid tag throughthe action of the ribozyme. The resulting polypeptide-nucleic acid tagconjugate comprises: the polypeptide, the peptide substrate of theribozyme used, a ribozyme RNA and the nucleic acid tag. In oneembodiment, a polypeptide-nucleic acid tag conjugate comprises apolypeptide-nucleic acid tag conjugate comprising: (a) a polypeptide ofinterest; (b) a peptide tag linked to the polypeptide of interest,wherein the peptide tag is the substrate of a ribozyme; (c) a ribozymeor a segment of a ribozyme for which the peptide tag of (b) is asubstrate, wherein the ribozyme or ribozyme segment is covalently linkedto the peptide tag; and (d) a nucleic acid tag, wherein the nucleic acidtag is linked to the ribozyme or ribozyme segment. In one embodiment,the peptide is linked to the 5' terminus of the ribozyme RNA and thenucleic acid tag extends from the 3' terminus of the ribozyme RNA. Thepeptide substrate of the ribozyme is referred to herein as the peptidetag. As a result of the specific interaction of the peptide tag on thepolypeptide with the ribozyme for which the peptide tag is a substrate,the polypeptide component is covalently linked to the nucleic acid tag.This technique has several clear advantages over other methods oftagging proteins. For example, because the peptide tag and the RNAcomponent of the ribozyme interact specifically with each other, theribozyme tagging reaction can be performed in a mixture of biologicalmolecules, such as in a crude cell lysate, as well as in vivo. Inaddition, because there is no limit on the number of different possiblenucleic acid tags, an essentially unlimited number of different(uniquely or specifically) tagged polypeptides can be produced. Onceproduced, the tagged proteins can be mixed and then detectedsimultaneously, using, for example, hybridization to DNA arrays.

Another embodiment of the present invention is a method of producing apolypeptide-nucleic acid tag conjugate, comprising: (a) combining: (1) apolypeptide of interest which bears a peptide tag wherein the peptidetag is a substrate for ribozyme RNA; (2) ribozyme RNA, wherein theribozyme RNA specifically covalently links to the peptide tag of (a)(1)in the presence of the remainder of the ribozyme and has linked theretoa nucleic acid tag; (3) the remainder of the ribozyme, thereby producinga combination; and (b) maintaining the combination under conditionsappropriate for ribozyme RNA of (a)(2) to associate with ribozyme RNA of(a)(3), forming a functional ribozyme that specifically covalently linksthe peptide tag to ribozyme RNA of (a)(2), whereby thepolypeptide-nucleic acid tag conjugate is produced.

Another embodiment of the present invention is a method of separating apolypeptide from a mixture of polypeptides, comprising: (a) tagging thepolypeptide at its amino terminus with a peptide tag, thereby producinga polypeptide-peptide tag, wherein the peptide tag is a substrate forribozyme RNA, thereby producing a mixture comprising thepolypeptide-peptide tag; (b) combining the mixture produced in (a) with(1) ribozyme RNA which bears a nucleic acid tag and specificallycovalently links to the peptide tag in the presence of the remainder ofthe ribozyme and (2) the remainder of the ribozyme, under conditionsappropriate for ribozyme (b) (1) to specifically interact with itspeptide substrate thereby forming a covalent link with the peptide andtagging the polypeptide with the nucleic acid tag, whereby a mixturecomprising a polypeptide-nucleic acid tag conjugate is formed; (c):combining the mixture formed in (b) with a nucleic acid which is abinding partner for the nucleic acid tag of the polypeptide-nucleic acidtag conjugate, whereby the binding partner hybridizes with the nucleicacid tag of the conjugate, forming a polypeptide-nucleic acid tagconjugate with the binding partner bound thereto; and (d) separating thepolypeptide-nucleic acid tag conjugate with the binding partner boundthereto from the product of (c), whereby the polypeptide is separatedfrom the mixture of polypeptides. In one embodiment, the binding partneris bound to a solid surface.

The present invention also relates to a method of detecting apolypeptide of interest in a mixture, wherein the method comprises: (a)combining a mixture to be assessed for the presence of thepolypeptide-nucleic acid tag conjugate with a binding partner for thenucleic acid tag, wherein the binding partner (1) is a nucleic acidsequence sufficiently complementary to the nucleic acid tag that thebinding partner and the nucleic acid tag of the conjugate bind to oneanother and remain bound under the conditions used and (2) is bound to asolid surface and wherein the combining occurs under conditionsappropriate for binding of the nucleic acid tag and the binding partnerand (b) detecting whether binding of the nucleic acid tag and thebinding partner occurred, wherein if binding is detected, thepolypeptide of interest is detected. In one embodiment, the bindingpartner or the nucleic acid tag is labeled with a detectable moiety(e.g., a chemical moiety, radioactivity) and detection of binding iscarried out by detecting the presence of the detectable moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an engineered mRNA. P-P-Prepresents the 5' triphosphate of the engineered mRNA. This triphosphatecan be modified or replaced to facilitate linkage to the translationproduct or to enhance the efficiency of in vitro translation; forexample, a 7-methyl guanosine cap can be used to increase the efficiencyof translation in eucaryotic translation extracts (e.g., those fromwheat germ extract or rabbit reticulocytes).

FIG. 2 is a schematic representation of an engineered mRNA in which theribozyme sequence includes a modified BIV-1 TAR RNA motif and the codingregion for a peptide tag encodes the Tat tag peptide (SEQ ID NO.: 1), orthe Tat 2 tag peptide (SEQ ID NO.: 2) where Tat is the BIV-1 Tat peptide(SEQ ID NO.: 3).

FIGS. 3A-3D represent schematically one embodiment of the invention inwhich a ribozyme comprised of two noncontiguous components is used toproduce polypeptide-engineered mRNA conjugates, and a representativeconjugate produced by the method. In FIG. 3A, initiation of translationon engineered mRNA is represented; the linear cross-hatched arearepresents a ribozyme segment which specifically covalently links to thepeptide tag. In FIG. 3B, presentation of the peptide tag and interactionof the two ribozyme segments is shown schematically; the L-shapedcross-hatched area represents the remaining segment of the ribozyme,which is bound to the ribozyme RNA which specifically covalently linksto the peptide tag. In FIG. 3C, binding of the peptide tag to theribozyme and the reaction covalently linking the peptide tag to theribozyme segment are represented. In FIG. 3D, the resulting encodedpolypeptide is represented schematically. The cross-hatched arearepresents ribozyme RNA.

FIG. 4 is a schematic representation of a ribozyme selection procedure.

FIG. 5A is an example of a ribozyme sequence (SEQ ID NO.: 5) thatattaches itself to the modified BIV Tat peptide. Upper case lettersrepresent the residues of the modified TAR RNA motif. This ribozyme wasisolated using the scheme shown in FIG. 4.

FIG. 5B is an example of a ribozyme sequence (SEQ ID NO.:6) thatattaches itself to the modified BIV-1 Tat peptide. Upper case lettersrepresent the residues of the modified TAR RNA motif.

FIG. 6 is an example of a two-part ribozyme in which an 18-nt ribozymesegment (SEQ ID NO.: 7) is sufficient to specifically covalently link tothe Tat tag peptide in the presence of the remainder of the ribozymesequence. Also shown is the 79-nt ribozyme segment (SEQ ID NO.: 8) whichis the second component of the two-part ribozyme. A model of thesecondary structure of the two-part ribozyme is shown.

FIG. 7 is a graphic representation of results of analysis, using aphosphorimager, of accumulation of the product of incubation of thetwo-part ribozyme shown in FIG. 6 with biotinylated Tat tag peptide.

FIGS. 8A-8C represent schematically a second embodiment of theinvention, by which polypeptide-nucleic acid tag conjugates areproduced. FIG. 8A depicts a polypeptide (with a peptide tag at its aminoterminus) which is to be tagged with a nucleic acid and a two-partribozyme, in which the portion of the ribozyme that becomes joined tothe polypeptide is joined to (has been extended by) a nucleic acid tagto be linked to the polypeptide. In FIG. 8B, the three componentsrepresented in FIG. 8A are combined and incubated under conditionsappropriate for function of the ribozyme used and, as illustrated, theribozyme specifically recognizes the amino terminal sequence (peptidetag) of the polypeptide and attaches the nucleic acid tag to the peptidetag. FIG. 8C shows that, because there is no limit to the number ofdifferent nucleic acid tags that can be used, many differentpolypeptides can be linked to nucleic acid tags and the resulting taggedpolypeptides then mixed together for simultaneous use.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a ribozyme which joins a polypeptide toa nucleic acid, products resulting from the joining activity of theribozyme, methods of making the products and uses therefor. As describedherein, the present invention can be seen as relating to two embodimentsin which the ribozymes of the present invention are used: one in whichthe ribozyme functions to join an mRNA to its translation product and isuseful to produce diverse libraries of encoded polypeptides and one inwhich the ribozyme functions to join a polypeptide of interest to anucleic acid which acts as a tag, producing polypeptide ofinterest-nucleic acid tag conjugates, in each of which the nucleic acidtag is different (specific), thus permitting identification, detection,separation, isolation, purification and/or alteration of characteristicsof the polypeptide of interest. Ribozymes of the present invention, aswell as the two embodiments, are described in detail herein and alsorepresented in the figures.

Ribozymes

Isolated ribozymes of the present invention are catalytically active;they interact with and bind their respective peptide substrate(s), whichin the embodiments described herein, act as peptide tags which, ineffect, act as links between the components joined by the ribozyme'sactivity. Isolated ribozymes of the present invention can function as asingle (contiguous) molecule or can occur as two ribozyme segments orcomponents, which come together, join or interact, under the conditionsused, to function as a complete ribozyme. The two components are onewhich comprises the ribozyme segment (or ribozyme RNA) whichspecifically interacts with (covalently links to) its peptide substrateand one which comprises the remainder of the ribozyme sequence.Ribozymes of this invention can comprise a motif or segment whichspecifically interacts with a peptide (its peptide substrate(s)) and isable to form a covalent bond with the peptide. For example, the isolatedribozymes represented schematically in FIG. 5A and FIG. 5B are specificexamples of ribozymes of the present invention. Another example of anisolated ribozyme of the present invention, which is a two-partribozyme, is represented in FIG. 6. The two-part ribozyme shown in FIG.6 includes ribozyme RNA sufficient to covalently link to a peptidesubstrate. In this embodiment, the ribozyme RNA sufficient to covalentlylink to the peptide tag is 18 nucleotides and links to the Tat tagpeptide or the Tat 2 tag peptide in the presence of the remainder of theribozyme. In this embodiment, the remainder of the ribozyme is 79nucleotides. It is to be understood that a wide variety of ribozymes andribozyme segments or portions are useful in producing the encodedpolypeptides and conjugates described herein. For example, sequencesrepresented herein can be altered by replacing, deleting, substituting,adding and/or modifying one or more nucleotides to produceribozymes/ribozyme segments which catalyze the same reaction (formationof a covalent bond with a peptide substrate). Altered sequences can beproduced using known methods and then assessed for their catalyticactivity, also using known methods. For example, the ribozyme sequencesrepresented in FIG. 5A or FIG. 5B can be altered by replacing, deleting,substituting, adding and/or modifying at least one nucleotide, providedthat they exhibit the ability to form a covalent link with a peptidesubstrate. Alteration can be extensive (e.g., 40% or more of thenucleotides can be altered). This is also the case for the two-partribozyme represented in FIG. 6, provided that the altered form of theribozyme RNA which specifically interacts with the peptide substrateretains or exhibits the ability to do so in the presence of theremainder of the ribozyme (which can be as shown in FIG. 6 or can itselfalso be altered in sequence). Alternatively, the sequence of theremainder of the ribozyme (e.g., the 79nt represented in FIG. 6 oranother sequence) can be altered. Altered ribozymes and ribozymesegments can include modified or artificial/non-naturally occurringnucleotides.

Isolated ribozymes described herein are useful, for example, to join anmRNA to its translation product and, thus, to produce diverse librariesof encoded polypeptides, as well as to tag polypeptides with specificnucleic acids, to produce polypeptide-nucleic acid tag conjugatesuseful, for example, for detection, identification, separation,isolation or purification of the polypeptide and to changecharacteristics of the polypeptide.

Described herein and represented in the figures are embodiments of thepresent invention in which a ribozyme functions to join a polypeptide toa nucleic acid through or by means of specific interaction of theribozyme with its substrate.

Generation of Diverse Libraries of Encoded Polypeptides

The present invention relates to a method of generating a diverselibrary or collection of encoded polypeptides; engineered mRNA fromwhich the diverse libraries of encoded polypeptides are produced;isolated ribozymes which join an mRNA to its translation product;encoded polypeptide libraries generated by the method;polypeptide-engineered mRNA conjugates of which the diverse encodedpolypeptide libraries are comprised; translation products of theengineered mRNAs; a method of identifying and, optionally, amplifyingmembers of the encoded polypeptide library (referred to as targetmembers) which have desired characteristics; target members of thelibrary identified by the method; polypeptide fragments of the targetmembers identified by this method; and ribonucleoprotein fragments ofthe target members identified by the method.

Engineered mRNA is used to produce a diverse encoded polypeptide libraryof the present invention; libraries of engineered mRNAs are oneembodiment of this invention. Engineered mRNA comprises: (a) ribozymeRNA which specifically covalently links to a peptide; (b) a codingregion for the peptide with which the ribozyme RNA specificallycovalently links (the peptide tag); (c) a region which encodes a diversepolypeptide and (d) two PCR primer-binding sites. The ribozyme RNAsegment which specifically covalently links to the peptide tag ispresent in (is part of) a ribozyme which occurs as a contiguous sequenceor is comprised of two noncontiguous components: one which comprises theribozyme RNA which specifically covalently links to the peptide tag andone which comprises the remainder of the ribozyme sequence. The twocomponents join together, under the conditions used to produce diverselibraries, to form a complete ribozyme. The region which encodes adiverse polypeptide differs among engineered mRNAs, thus making itpossible to produce a diverse encoded polypeptide library.

In addition to the elements described in the previous paragraph,engineered RNA can include one or more of the following optionalcomponents: three phosphate groups at the 5' end of the ribozyme RNA oran mRNA cap, such as a 7-methyl guanosine triphosphate; an RNA linkerbetween the ribozyme sequence and the peptide tag coding region; acoding region for a peptide linker between the coding region for thepeptide tag and the diverse coding region and a ribosomal stalling sitebetween the diverse polypeptide segment and the second PCRprimer-binding site.

The coding regions for the peptide tag, the optional peptide linker, andthe diverse polypeptide segment are all fused in-frame so that they aretranslated as a single tripartite polypeptide. All components encoded bythe engineered mRNA can be immediately adjacent to one another or can beseparated from the preceding and/or following component(s) by anintervening amino acid residue or intervening amino acid residues.Similarly, components of the engineered mRNA can be separated byintervening nucleotides, provided that the components of the openreading frame remain in frame.

The ribozyme sequence of the engineered mRNA comprises a motif (aregion) that interacts specifically with the peptide tag, with theresult that polypeptide-engineered mRNA conjugates are produced. Theribozyme promotes the formation of a covalent bond between the ribozymeRNA (e.g., the 5'end) of the engineered message and a reactive group,such as a hydroxyl group or an amino group, of the peptide tag. Theregion coding for a peptide linker is an optional component of theengineered mRNA; however, if a peptide linker is to be produced, it mustbe of appropriate length (e.g., 2-10, 10-20, 15-25 or 20-30 amino acidresidues and typically 30 amino acid residues or more) and compositionto allow for presentation of the peptide tag for formation of thecovalent bond. In an alternative embodiment, part (or all) of theribozyme can reside within or downstream of the mRNA coding region. Inanother alternative embodiment, the peptide tag can be C-terminal of thediverse polypeptide segment and the engineered mRNA is designedaccordingly.

In one embodiment, the engineered mRNA includes, in order (from the 5'end to the 3' end of the mRNA): a first PCR primer-binding site; a novelcatalytic RNA (ribozyme) sequence, which may include or overlap with thePCR primer binding site; a coding region for a peptide tag whichinteracts specifically with a motif in the ribozyme sequence;optionally, a region coding for a peptide linker; a coding region(referred to as the diverse coding region) that differs among members ofthe library and encodes a polypeptide (referred to as the diversepolypeptide segment); and a second primer-binding site for reversetranscription and PCR amplification.

In a specific embodiment of the present invention, the engineered mRNAcomprises (5'→3'): a novel ribozyme which comprises a motif, modifiedBIV-1 TAR RNA, which interacts specifically with the translated productof the second component of the engineered mRNA (such as that shown inFIG. 5), a peptide tag; a coding region for the peptide Tat tag (SEQ IDNO.: 1) or the Tat 2 tag peptide (SEQ ID NO.:2), which includes theBIV-1 (Bovine Immunodeficiency Virus-1) Tat peptide (SEQ ID NO.: 3); acoding region for a linker sequence of sufficient length (typically 30amino acid residues or more) to allow presentation of the peptide tag topermit it to interact with the ribozyme; and a diverse coding regionencoding the diverse polypeptide segment. In this embodiment, theengineered mRNA also comprises: a first PCR primer-binding site, locatedwithin the ribozyme sequence and overlapping the 5' portion of themodified BIV-1 TAR motif, and a second PCR primer-binding site, located3' of the coding region.

Isolated ribozymes, which are catalytically active and assume aconformation which permits them to join or link an mRNA to thetranslation product of the mRNA, are also the subject of this invention.Such isolated ribozymes can be a contiguous molecule or can occur as tworibozyme segments or components, which can come together or interact toform the complete ribozyme. The two components are one which comprisesthe ribozyme RNA which specifically covalently links to the peptide tagand one which comprises the remainder of the ribozyme sequence. Isolatedribozymes of this invention comprise a motif which specificallyinteracts with a peptide and are able to form a covalent bond betweenthemselves and the peptide. For example, when such isolated ribozymesare incorporated in (are a component of) an engineered mRNA of thepresent invention and the engineered mRNA undergoes translation asdescribed herein, they join or link the engineered mRNA to itstranslation product by promoting formation of a covalent bond betweenthe ribozyme RNA, the engineered message and a reactive group of thepeptide tag encoded by the engineered mRNA. Specific examples ofisolated ribozymes are those represented by SEQ ID NO.: 5 and SEQ IDNO.: 6, which are represented schematically in FIGS. 5A and 5B. Theconformations shown in FIGS. 5A and 5B are secondary structures it isreasonable to propose for the ribozyme of SEQ ID NO.: 5 and SEQ ID NO.:6, respectively, but Applicants are not intending to be bound by thishypothesis. Another example of an isolated ribozyme is that representedin FIG. 6. This is an example of a two-part ribozyme, as describedbelow. Other isolated ribozymes which comprise a nucleotide (RNA)sequence sufficiently similar to that of SEQ ID NO.: 5 or 6 and join anmRNA to the translation product of the mRNA, such as an isolatedribozyme which comprises a nucleotide sequence sufficiently similar toSEQ ID NO.: 5 or 6 that it results in the active (functional)conformation of the ribozyme of SEQ ID NO.: 5 or 6 (it has importantsecondary structural features assumed by the ribozyme of SEQ ID NO.: 5or 6), are also the subject of this invention.

In another embodiment of the present invention, only a portion orsegment (one or more nucleotides) of the ribozyme resides within the 5'untranslated region of the engineered mRNA; the remainder of theribozyme resides elsewhere (e.g. within or downstream of the codingregion, or on a separate RNA molecule). Although the parts or componentsof the ribozyme are not contained within a single contiguous sequence,they come together to form a ribozyme complex which is functionallyequivalent to the ribozyme of the previously described embodiment(functionally equivalent to a complete ribozyme). For example, one ormore nucleotides (e.g., the first nucleotide, the first 13, 14, 15, 16,17 or 18 nucleotides of the ribozyme, or more) are at the 5' end of theengineered mRNA; the remaining residues are supplied as a separate RNAmolecule such that a ribozyme complex is formed and binds the tagpeptide and covalently joins the ribozyme portion or segment to thepeptide tag. As used herein, the term "ribozyme RNA" includes ribozymes,ribozyme portions and ribozyme segments.

In an example of this embodiment, pppGGA, the first three residues ofthe ribozyme shown in FIG. 5 (SEQ ID NO.: 5), resides at the 5' end ofthe engineered mRNA and the remaining ribozyme residues are supplied asa separate RNA molecule such that a ribozyme complex is formed thatbinds the Tat tag peptide and covalently joins the GGA ribozyme segmentto the peptide tag. In a further example of this embodiment, as many asthe first 18 residues of the ribozyme shown in FIG. 5 (e.g., fromresidues 1 to 18 and any shorter portions such as the first 13, 14, 15,16 or 17 residues) reside at the 5' end of the engineered mRNA, and theremaining ribozyme residues are supplied as a separate RNA molecule suchthat the two components form a ribozyme complex (complete ribozyme) thatbinds the Tat tag peptide and covalently joins the ribozyme segment tothe peptide tag. One example of an 18 nucleotide (nt) ribozyme RNAsegment (GGACAGCUCCGAGUGUCC; SEQ ID NO.: 7) that specifically covalentlylinks to the Tat tag peptide in the presence of the remainder of theribozyme sequence is shown in FIG. 6. In another embodiment, theribozyme RNA that specifically covalently links with a peptide is atleast 18 nucleotides of a ribozyme or is a contiguous sequence.

In the method of generating libraries of encoded polypeptides,engineered mRNAs and sufficient in vitro translation mixture arecombined to produce a combination of the two and the combination ismaintained under conditions appropriate for translation of theengineered mRNAs (and, thus, expression of the product encoded by theengineered mRNAs) to occur. In one embodiment, a library of encodedpolypeptides is produced by combining an in vitro translation mixturederived from rabbit reticulocytes and a library of engineered mRNAs,described herein, in which a ribozyme (such as that shown in FIG. 5)includes a modified BIV-1 TAR RNA motif and in which the coding regionfor a peptide tag encodes the peptide Tat tag peptide (SEQ ID NO.: 1).The in vitro translation mixture is a combination of biological reagentsand cellular components which translates the engineered mRNA to producethe polypeptide product it encodes. In vitro translation mixtures fromE. coli, wheat germ and rabbit reticulocytes are commercially availablefrom Promega.

When the in vitro translation mixture and a library of engineered mRNAare combined and maintained under appropriate conditions, translation isinitiated at the start codon (e.g., AUG) of the engineered mRNA andproceeds 5'→3'. The peptide tag is produced, followed by the linker, ifone is encoded by the engineered mRNA. After the peptide tag emergesfrom a translating ribosomal complex, the ribozyme interacts with thepeptide tag and promotes formation of a covalent linkage between theribozyme RNA of the message and a reactive group, such as a hydroxylgroup or an amino group, of the peptide tag. If a linker is encoded bythe engineered mRNA, the linker permits the peptide tag to be presentedin such a manner that the reactive group of the peptide tag is availablefor formation of the covalent linkage with the 5' end of the engineeredmRNA. Translation of the diverse coding region can occur before, during,or after the covalent linkage of the peptide tag and an engineered mRNA.As a result, the newly-produced polypeptide is covalently linked to theengineered mRNA that encodes it.

Encoded polypeptides produced by the method described herein andlibraries of encoded polypeptides are also the subject of thisinvention. The encoded polypeptides are conjugates which each, in turn,comprise an engineered mRNA, linked to its translation product by acovalent bond between the 5' end of the engineered mRNA and a reactivegroup, such as a hydroxyl group or an amino group, of the peptide tag(the ribozyme substrate). The translation product of an engineered mRNAcomprises the peptide tag and the diverse polypeptide segment and,optionally, the linker sequence. If a linker sequence is present, theterm translation product refers to these three components. Translationproducts are also the subject of this invention.

Thus, encoded polypeptides of the present invention, which are describedabove with reference to the engineered mRNAs which encode them, eachcomprise an engineered mRNA and its translation product, joined by acovalent bond between the ribozyme RNA of the engineered mRNA and acomponent of the translation product (the peptide tag). The peptide tagcan be any peptide of appropriate composition and length to result inspecific interaction between itself and the ribozyme sequence. In oneembodiment, the peptide tag is the Tat tag peptide (SEQ ID NO.: 1),which includes the BIV-1 Tat peptide. Alternatively, the peptide tag isthe Tat 2 tag peptide (SEQ ID NO.:2).

The optional peptide linker is of sufficient length and composition forit to present the peptide tag for formation of a covalent bond between areactive group of the tag and the ribozyme RNA of the engineered mRNA.That is, the peptide tag and the ribozyme RNA of the engineered mRNAmust come together such that the covalent bond is formed.

The diverse polypeptide segment can be a polypeptide of any length andcan be encoded by mRNA sequences which occur in nature (such as in alibrary which represents the mRNA or DNA sequences present in cells) oris produced chemically. For example, the mRNA or DNA present inprokaryotic or eukaryotic cells (including invertebrate and vertebratecells, including mammalian, such as human cells) can be used to producea library of engineered mRNAs, which, by in vitro translation, result inproduction of a library of encoded polypeptides in which the diversepolypeptide segments represent the population of polypeptides expressedin the cells. Alternatively, randomly-produced mRNA, such as thatproduced by combinatorial chemical synthesis to produce mRNAs or bytranscribing DNAs produced by combinatorial chemical synthesis of DNA,can be used to make engineered mRNA libraries.

The components of the translation product can be immediately adjacent toone another or can be separated by one or more intervening amino acidresidues (amino acid residues which are not members of the peptide tag,peptide linker or diverse polypeptide segment).

In one embodiment, a diverse library of encoded polypeptides iscomprised of conjugates of an engineered mRNA and its translationproduct in which the ribozyme, such as that shown in FIG. 5, includes amodified BIV-1 TAR RNA motif (SEQ ID NO.:3) and the coding region for apeptide tag encodes the Tat tag peptide (SEQ ID NO.: 1) or the BIV-1 Tat2 tag peptide (SEQ ID NO.: 2).

Once a diverse encoded polypeptide library has been produced, it can bescreened, using known methods, to identify target members which areconjugates with desired characteristics. A key advantage of the diverseencoded polypeptide library of the present invention, which is comprisedof polypeptide-engineered mRNA conjugates, is that even members whichoccur in small numbers (rare members) and are of interest because ofdesired biological or biochemical properties (e.g., binding to aparticular ligand, enzymatic activity) can be enriched and thenidentified by amplification, cloning and sequencing of their respectivemRNAs.

A diverse library of encoded polypeptides can be enriched in targetmembers, using known methods. Methods by which target members of thelibrary can be enriched and identified include affinity enrichment usingimmobilized ligand or binding partner and, for enzymatic activity,affinity to a product of a reaction in which the enzyme has modifieditself (with, for example, a mechanism-based inhibitor) or a substrateto which it is attached (see, e.g., Williams, K. P. and D. P. Bartel,"In Vitro Selection of Catalytic RNA", pp. 367-381 In: Catalytic RNA,(Fritz Eckstein and David M. J. Lilley, Ed.), Springer (1996)).

Furthermore, libraries enriched in target members can be amplified andsubjected to additional enrichment and amplification. For example, alibrary of conjugates that has been enriched for a desired activity (anenriched encoded polypeptide library) can be reverse transcribed,producing the cDNA of the mRNA components. The cDNAs can then beamplified (e.g., by PCR or other amplification methods). The resultingPCR products are subjected to in vitro transcription, resulting inproduction of an amplified pool of engineered mRNAs of the enrichedconjugate library. In vitro translation of this pool results in linkingof the mRNA to its translation product, producing an amplified versionof the enriched encoded polypeptide library. Conjugates amplified inthis way are subjected to further enrichment and amplification, which isrepeated as necessary until target members are enriched to the desiredextent (e.g., enriched to a level where they are present in sufficientnumbers to be detected by binding to a ligand of interest, or catalyzinga reaction of interest). After sufficient enrichment, mRNA targetmembers are cloned and individual conjugates can be screened for thedesired function. The translation product of the mRNA or a fragment ofthe translation product (particularly, the diverse polypeptide segment)can also be screened for activity without attachment to the engineeredmRNA. One embodiment of the present method of identifying members of adiverse encoded polypeptide library which exhibit a desired activity orcharacteristic (target members) comprises the steps of:

(a) producing a diverse encoded polypeptide library which comprisespolypeptide-engineered mRNA conjugates by:

(i) combining:

(1) a library of engineered mRNAs, each of which comprises, in order(from the 5' end of the mRNA to the 3' end of the mRNA): a) a ribozymesequence which comprises a first PCR primer--binding site and a motifthat interacts specifically with a peptide; b) a coding region for thepeptide (referred to as a peptide tag) with which the motif of theribozyme sequence interacts specifically; c) a region coding for alinker sequence; d) a coding region for a diverse polypeptide segment;and e) a second PCR primer-binding site and

(2) an appropriate in vitro translation mixture, with the result that acombination is produced;

(ii) maintaining the combination under conditions appropriate fortranslation of the engineered mRNA to occur, whereby the translationproduct of the engineered mRNA (which comprises the peptide tag, thelinker sequence and the diverse polypeptide segment) is produced andjoined to the engineered mRNA by a covalent bond between the 5' end ofthe engineered mRNA and a reactive group of the peptide tag, therebyproducing polypeptide-engineered mRNA conjugates;

(b) enriching the diverse encoded polypeptide library for members whichexhibit a desired activity, thereby producing an enriched polypeptidelibrary comprised of polypeptide-engineered mRNA conjugates;

(c) amplifying the enriched polypeptide library by:

(i) reverse transcribing the engineered mRNA component of theconjugates, thereby producing the corresponding cDNA,

(ii) amplifying and transcribing in vitro the corresponding cDNA,thereby producing a pool of amplified, enriched engineered mRNA;

(iii) combining the pool of amplified, enriched engineered mRNA with anappropriate in vitro translation mixture, thereby producing acombination;

(iv) maintaining the combination under conditions appropriate fortranslation of the engineered mRNA to occur, whereby the translationproduct of the engineered mRNA is produced and joined to the engineeredmRNA by a covalent bond between the 5' end of the engineered mRNA and areactive group of the peptide tag, thereby producing an amplifiedenriched polypeptide library comprised of polypeptide-engineered mRNAconjugates;

(d) repeating steps b) and c), as necessary, until members which exhibitthe desired activity are present in sufficient number to be detected;and

(e) detecting members which exhibit the desired activity,

thereby identifying members which exhibit the desired activity. In oneembodiment of the method, the ribozyme sequence comprises SEQ ID NO.: 5.In another, the motif that interacts specifically with a peptide ismodified BIV-1 TAR RNA and the peptide tag is the Tat tag or the Tat 2tag peptide; the ribozyme sequence in this embodiment can comprise SEQID NO.: 5. In the method, the region coding for a linker sequenceencodes a linker of appropriate length, as described previously;typically it encodes a linker of 30 amino acid residues or more,although a shorter linker (e.g., 10 or more amino acid residues) can beused.

The translation products of the enriched mRNA (the polypeptide componentof the target members), such as polypeptides which display activities ofinterest (e.g., ligand binding or catalytic activity) as well asengineered derivatives of these translation products which displayactivities of interest are referred to as target polypeptide fragments.These target polypeptide fragments are also the subject of thisinvention. Target polypeptide fragments can be released or separatedfrom target members in which they occur, using known methods (e.g.,RNA), or they can be synthesized without attachment to the mRNA (e.g.,using chemical synthesis or mRNA translation in the absence of the tRNAanalogue. They can be used, for example, as diagnostic or therapeuticreagents, (e.g., single-chain monoclonal antibodies), protein catalysts,members of binding pairs, receptors or their ligands, enzymes or enzymesubstrates. Once a polypeptide fragment which has desiredcharacteristics has been identified, it can be produced using knownmethods (e.g., production in an appropriate expression system, chemicalsynthesis).

Ribonucleoprotein fragments of the target members are also the subjectof this invention. They can be used, for example, as enzymes or ligands.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

Tagging Polypeptides with Nucleic Acids

Purification or detection of nucleic acids is typically more facile thanis purification or detection of proteins. This is an inherent advantageof nucleic acids, which results from the predictable hybridizationproperties of nucleic acid target sequences. Nucleic acids with highaffinity and specificity for the target sequence(s) can readily bedesigned and synthesized. In addition, large numbers of nucleic acidsequences can be analyzed in parallel using, for example, hybridizationto DNA chips. In contrast, detecting a particular protein target oftenrequires investing considerable effort in generating antibodies oraptamers that specifically recognize the target protein. Although thisdifficulty in detecting and purifying can be overcome for recombinantproteins by using affinity tag fusions, the parallel detection of largenumbers of different recombinant proteins is limited by the smallrepertoire of affinity tags, as well as distinct--and sometimesincompatable--incubation protocols for each tag. This limitation can beovercome by attaching nucleic acid tags to polypeptides (also referredto as proteins); as described herein, tagging polypeptides isaccomplished through the activity of isolated ribozymes of the presentinvention, which specifically recognize and bind to a peptidesubstrate(s). Polypeptides tagged with a specific or selected nucleicacid (RNA or DNA) are referred to herein as polypeptide-nucleic acid tagconjugates and comprise: a polypeptide of interest (or targetpolypeptide); a peptide substrate of the ribozyme used to join thepolypeptide and the nucleic acid components (also referred to as apeptide tag); ribozyme RNA and a nucleic acid tag. Polypeptide-nucleicacid conjugates, methods of producing the conjugates, methods of usingthe conjugates are also the subject of this invention.

Polypeptide-nucleic acid tag conjugates are produced as follows: Apolypeptide of interest which is fused with a peptide tag which is asubstrate of the ribozyme is combined with (a) ribozyme RNA whichspecifically covalently links to the peptide tag and has linked theretoor bears a nucleic acid tag and (b) additional ribozyme RNA which, incombination with segment bearing the nucleic acid tag forms acomplete/functional ribozyme, under conditions appropriate for theribozyme RNA of (a) to specifically covalently link to the peptide tag.As a result, the polypeptide of interest is tagged with the nucleic acidtag. Alternatively, a complete ribozyme bearing a nucleic acid tag iscombined with the polypeptide of interest bearing the peptide tag(ribozyme substrate). For example, a polypeptide of interest which isfused at its amino terminus with a peptide tag which is a peptidesubstrate of the ribozyme to be used is combined with: (a) the ribozyme,which bears or is joined with a specific nucleic acid sequence (thenucleic acid tag), or (b) segments of a ribozyme, wherein one ribozymesegment is joined at its 3' end with a nucleic acid sequence and in thepresence of a second ribozyme segment forms a covalent bond with theamino terminus of the peptide tag under conditions appropriate for theribozyme RNA to specifically interact with its peptide substrate (thepeptide tag present on the polypeptide of interest). As a result, theribozyme or ribozyme segment (which bears the specific nucleic acidsequence) interacts specifically with the peptide tag of the polypeptideof interest, forming a covalent link or bond and, thus, tagging thepolypeptide of interest with the specific nucleic acid. FIGS. 8A-8Crepresent a specific embodiment of this process schematically. In theembodiment represented in FIG. 8A, a polypeptide is tagged at its aminoterminus with a peptide tag which is a substrate for the ribozyme to beused in producing polypeptide-nucleic acid conjugates. The peptide tagis joined or linked to the amino terminus of the polypeptide using knownrecombinant methods. The polypeptide can be any polypeptide it isdesired to detect, identify, isolate, purify or alter in terms of itscharacteristics (e.g., solubility, size, functions, intracellularlocation). It can be of any length and can be present, for example, in abody fluid or tissue, cell lysate, food product, beverage, water, orlibrary of proteins. Also shown in the example as represented in FIG.8A, the ribozyme to be used can comprise two components: a first region,which recognizes or becomes joined to the peptide tag on thepolypeptide, which bears the nucleic acid tag at its 3' end and a secondregion, which is the remainder of the ribozyme (which, when interactingwith the first ribozyme component, forms a complete/functionalribozyme). In FIG. 8A, P-P-P represents the 5' triphosphate of theribozyme of the specific embodiment shown.

As shown in FIG. 8B, when the three entities represented in FIG. 8A arecombined under appropriate conditions (e.g., 1 hr. in 100 mM KOAc, 50 mMTris-OAc pH7.5, 30 mM NH₄ OAc, 15 mM Mg(OAc)₂, 2 mM DTT, a functionalribozyme is formed and the polypeptide is joined to the nucleic acid tagthrough the interaction of the ribozyme (which bears the nucleic acidtag) with the peptide tag (ribozyme substrate). In a specificembodiment, in which the two-part ribozyme represented in FIG. 6 isused, the covalent linkage is formed by attack of the amino terminus ofthe peptide tag on the 5' triphosphate of the ribozyme, as indicated bythe arrow in FIG. 8B. FIG. 8C is a schematic representation of resultingpolypeptide-nucleic acid tag conjugates, which comprise the polypeptideof interest, the peptide tag (present at the amino terminus of thepolypeptide), the ribozyme covalently joined to the peptide tag and thenucleic acid tag. As discussed above, the variety of nucleic acid tagsis unlimited and, thus, an unlimited number of polypeptides can betagged with a specific (different) nucleic acid.

The ribozymes described above can be used for generating thepolypeptide-nucleic acid tag conjugates of this invention. The nucleicacid tags can be any sequence of nucleotides and can be DNA, RNA, PNA orany other type of modified nucleic acid. They can be made using knownmethods and can be of any length, provided that the tag remainshybridized with its complement (binding partner) under the conditionsused. They will generally be at least 10 nucleic acids in length andrange from 4 to 30 or more nucleic acids in length.

Polypeptide-nucleic acid tag conjugates have a wide variety of uses,which encompass essentially any context in which separation, isolation,purification, detection or identification of a recombinant polypeptideis desired and/or in which alteration of a characteristic(s) of thepolypeptide is desired. For example, in one embodiment, polypeptides ina cell lysate are tagged, as described herein, with a nucleic acid. Eachpolypeptide is preferably tagged with a different nucleic acid, with theresult that each polypeptide is in essence uniquely tagged. Thecomplementary sequence of a nucleic acid tag is used as a bindingpartner for the tag and, in one embodiment, is bound to a solid surface.Hybridization of the nucleic acid tag of the conjugate with itscomplement results in attachment of the complex to a solid surface(e.g., beads, chip or other planar surface, wells, columns) if thecomplement is bound to a solid surface and remains bound under theconditions used. Alternatively, the binding partner can be in solutionor suspension and hybridization, thus, occurs in the solution orsuspension. The binding partner can be totally complementary to thenucleic acid tag or can be less than totally complementary in sequence,provided that the nucleic acid tag of the conjugate remains bound(hybridized) to the binding partner nucleic acid under the conditionsused. Subsequently, the polypeptide-nucleic acid tag conjugate can bereleased from the binding partner by changing conditions such thathybridization no longer occurs (e.g., by lowering ionic strength.). Thepolypeptide can be separated from the complex, for example, forcharacterization (such as sequencing or structural assessments,modification or determination of biological activity/function) usingknown methods (e.g., nuclease treatment). Polypeptides obtained in thisway are useful as drugs, drug targets, and diagnostic reagents.

Alternatively, polypeptide-nucleic acid tag conjugates can be formed inorder to alter a characteristic or characteristics of polypeptides. Forexample, solubility of polypeptides can be altered [reduced or enhanced]in this manner. Increasing the solubility of an expressed protein byadding a nucleic acid tag can prevent aggregation during proteinpurification without disrupting the native fold of the protein. Activityof a polypeptide can also be altered, making it possible to expresslevels of the protein that would otherwise be toxic. Mobility of apolypeptide can be altered by tagging with a nucleic acid, providing ameans by which polypeptides in a mixture can be separated from oneanother, using, for example, gel electrophoresis. The invention isillustrated by the following examples, which are not intended to belimiting in any way.

EXEMPLIFICATIONS Example 1

A Ribozyme That Links a Peptide Tag to Itself

This ribozyme has the sequence:

GGACAGCUCCGAGCAUUCUCGUGUAGCUCUGACCAUGGAGUACAGCACCACGUCGUCGCUGGUAUAUGGCCAAGUAAUAAACGACUCAUCCCUCCAAG (SEQ ID NO.: 5)

Without wishing to be bound by theory, the ribozyme is predicted to foldinto the secondary structure shown in FIG. 5. In FIG. 5, the modifiedTAR motif is indicated (uppercase).

The 5' ribozyme activity was isolated from a large population of >10¹⁴randomized RNA molecules following the procedure outlined below andrepresented in FIG. 4.

1) In vitro transcribed RNA was incubated overnight with a biotinylatedTat tag peptide (MSYSGPRPRGTRGKGRRIRRGGK-BIOTIN).

2) The RNA population was reverse transcribed to generate an RNA-cDNAheteroduplex.

3) Ribozyme molecules that joined themselves to the biotinylated peptidewere separated from unreacted molecules by incubating the heteroduplexwith paramagnetic particles coated with Streptavidin.

4) The RNA strand of the heteroduplex was cleaved with alkali to freethe DNA copy of the RNA from the support.

5) This DNA was amplified using PCR.

6) The resulting double-stranded DNA was then used as template in an invitro transcription reaction to generate a new population of RNAmolecules. Repeating steps 1-6 selectively enriched for ribozymes thatcan covalently attach themselves to the modified BIV-1 peptide.

Example 2

An 18-nt Ribozyme RNA Segment that Specifically Covalently Links to theTat Tag Peptide in the Presence of the Remainder of the RibozymeSequence

16.9.split (FIG. 6) is a two-part ribozyme that is a version of theribozyme shown in FIG. 5A (SEQ ID NO: 5), where the ribozyme has beensplit at the loop of paired-region 2 (P2) and the P2 helix has beenextended by 5 base pairs. The loops of P5 and P6 have also beenshortened and stabalized. This 18-nt RNA (SEQ ID NO: 7,pppGGACAGCUCCGAGUGUCC) and 79-nt RNA (SEQ ID NO. 8,pppGGACACUCGUGUAGCUCUGACCAUGGAGUACAGCUUCGGCUGGUAUAUGGCCAAGUACUUCGGUACUCAUCCCUCCAAG) function together as a ribozyme inwhich the 18-nt ribozyme RNA segment specifically covalently links tothe Tat tag peptide.

The two ribozyme RNAs of 16.9.split (SEQ ID NO: 7 and 8) shown in FIG. 6were transcribed in vitro using T7 RNA polymerase and appropriatetemplates. The RNAs were gel purified, then incubated together withbiotinylated Tat tag peptide (SEQ ID NO.1) in reaction buffer 1.2 μMradiolabeled SEQ ID NO 4, 3.3 μM SEQ ID NO. 7, 1 mM Tat tag peptide, 100mM KOAc, 50 mM Tris-OAc pH 7.5, 30 mM NH₄ OAc, 15 mM Mg(OAc)₂, 2mM DTT).Aliquots were taken at 0, 1,2, and 4 hours and the accumulation ofproduct observed as a shifted product on a 15% polyacrylamide/8M ureadenaturing gel. The gel was analyzed using a phosphorimager and thefraction of labeled 18 nt ribozyme RNA that had been linked to thepeptide was determined (FIG. 7).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain, using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 8                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: BIV-1                                                          - - <400> SEQUENCE: 1                                                         - - Met Ser Tyr Ser Gly Pro Arg Pro Arg Gly Th - #r Arg Gly Lys Gly Arg       1               5  - #                10  - #                15               - - Arg Ile Arg Arg Gly Gly Lys                                                          20                                                                 - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 23                                                              <212> TYPE: PRT                                                               <213> ORGANISM: BIV-1                                                          - - <400> SEQUENCE: 2                                                         - - Met Lys Tyr Ser Gly Pro Arg Pro Arg Gly Th - #r Arg Gly Lys Gly Arg       1               5  - #                10  - #                15               - - Arg Ile Arg Arg Gly Gly Lys                                                          20                                                                 - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 17                                                              <212> TYPE: PRT                                                               <213> ORGANISM: BIV-1                                                          - - <400> SEQUENCE: 3                                                         - - Ser Gly Pro Arg Pro Arg Gly Thr Arg Gly Ly - #s Gly Arg Arg Ile Arg       1               5  - #                10  - #                15               - - Arg                                                                       - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: BIV-1                                                          - - <400> SEQUENCE: 4                                                         - - ggacagcucc gagcauucuc guguagcu         - #                  - #                 28                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 98                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: ribozyme sequence                                     - - <400> SEQUENCE: 5                                                         - - ggacagcucc gagcauucuc guguagcucu gaccauggag uacagcacca cg -             #ucgucgcu     60                                                                 - - gguauauggc caaguaauaa acgacucauc ccuccaag      - #                      - #     98                                                                     - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 152                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Ribozyme sequence                                     - - <400> SEQUENCE: 6                                                         - - ggacagcucc gagcauucuc guguagcucu gaccaugggg uaggcuugaa uu -             #caguguuc     60                                                                 - - gacgauucac uaguuucgaa acauaaguca gccguauaua gucacuuugu ca -            #ggcgccac    120                                                                 - - cuaggucgga ccugacaaug caucccccca gg       - #                  - #             152                                                                     - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Ribozyme sequence                                     - - <400> SEQUENCE: 7                                                         - - ggacagcucc gagugucc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 79                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Ribozyme sequence                                     - - <400> SEQUENCE: 8                                                         - - ggacacucgu guagcucuga ccauggagua cagcuucggc ugguauaugg cc -             #aaguacuu     60                                                                 - - cgguacucau cccuccaag             - #                  - #                      - # 79                                                                __________________________________________________________________________

We claim:
 1. Engineered mRNA which comprises:(a) ribozyme RNA whichspecifically covalently links to a peptide in the presence of theremainder of the ribozyme; (b) a coding region for the peptide withwhich the ribozyme RNA of (a) specifically covalently links, wherein thepeptide is referred to as a peptide tag; (c) a coding region for adiverse polypeptide; and (d) two PCR primer-binding sites.
 2. Theengineered mRNA of claim 1 wherein the ribozyme RNA of (a) is onenucleotide.
 3. The engineered mRNA of claim 2 wherein the ribozyme RNAof (a) is guanosine triphosphate.
 4. The engineered mRNA of claim 1wherein the ribozyme RNA of (a) is from 1 to about 18 nucleotideresidues, inclusive, wherein the 1 to 18 nucleotide residues are fromthe 5' end of the ribozyme sequence.
 5. The engineered mRNA of claim 1wherein the ribozyme of (a) comprises at least 18 nucleotides.
 6. Theengineered mRNA of claim 1 wherein the ribozyme RNA of (a) is acontiguous sequence.
 7. The engineered mRNA of claim 1 wherein theribozyme comprises modified BIV-1 TAR RNA and the peptide tag is the Tattag peptide or the Tat 2 tag peptide.
 8. The engineered mRNA of claim 1which further comprises a coding region for a peptide linker, whereinthe coding region for the peptide linker is located between (b) and (c).9. The engineered mRNA of claim 8 wherein the region coding for a linkersequence encodes a linker of 30 amino acid residues or more.
 10. Theengineered mRNA of claim 1 which further comprises a ribosome stallingsite located between the coding region for a diverse polypeptide and thesecond PCR primer-binding site.
 11. The engineered mRNA of claim 1wherein the ribozyme is the ribozyme of SEQ. ID NO.: 5 or SEQ ID NO.:6.12. A library of engineered mRNAs wherein each engineered mRNA in thelibrary comprises:(a) ribozyme RNA which specifically covalently linksto a peptide in the presence of the remainder of the ribozyme; (b) acoding region for the peptide with which the ribozyme RNA of (a)specifically covalently links, wherein the peptide is referred to as apeptide tag; (c) a coding region for a diverse polypeptide segment; and(d) two PCR primer-binding sites.
 13. The library of claim 12 whereinthe ribozyme RNA of (a) is one nucleotide.
 14. The library of claim 13wherein the ribozyme RNA of (a) is guanosine triphosphate.
 15. Thelibrary of claim 12 wherein the ribozyme RNA of (a) is from 1 to about18 nucleotide residues, inclusive, wherein the 1 to about 18 nucleotidesare from the 5' end of the ribozyme sequence.
 16. The library of claim12 wherein the ribozyme RNA of (a) comprises at least 18 nucleotides.17. The library of claim 12 wherein the ribozyme RNA of (a) is acontiguous sequence.
 18. The library of claim 12 wherein the ribozymeRNA comprises modified BIV-1 TAR RNA and the peptide tag is the Tat tagpeptide or the Tat 2 tag peptide.
 19. The library of claim 12 whereineach engineered mRNA additionally comprises a coding region for apeptide linker, wherein the coding region for the peptide linker islocated between (b) and (c).
 20. The library of claim 19 wherein theregion coding for a peptide linker encodes a linker of 30 amino acidresidues or more.
 21. The library of claim 12 wherein the engineeredmRNA further comprises a ribosome stalling site located between thecoding region for a diverse polypeptide and the second PCRprimer-binding site.
 22. The library of claim 12 wherein in eachengineered mRNA the ribozyme RNA is the ribozyme of SEQ. ID No.: 5 orSEQ ID NO.:
 6. 23. A polypeptide-engineered mRNA conjugatecomprising:(a) an engineered mRNA which comprises:(1) ribozyme RNA whichspecifically covalently links to a peptide in the presence of theremainder of the ribozyme; (2) a coding region for the peptide withwhich the ribozyme RNA of (a) covalently links, wherein the peptide isreferred to as a peptide tag; (3) a coding region for a diversepolypeptide; and (4) two PCR primer-binding sites; and (b) thetranslation product of the engineered mRNA, which comprises the diversepolypeptide and the peptide tag,wherein the ribozyme RNA of theengineered mRNA is joined to the peptide tag by a covalent bond betweenthe ribozyme RNA of the engineered mRNA and a reactive group of thepeptide tag.
 24. The polypeptide-engineered mRNA conjugate of claim 23wherein the ribozyme RNA of (a) is one nucleotide.
 25. Thepolypeptide-engineered mRNA conjugate of claim 23 wherein the ribozymeRNA of (a) is from 1 to about 18 nucleotide residues, inclusive, whereinthe 1 to about 18 nucleotides are from the 5' end of the ribozymesequence.
 26. The polypeptide-engineered mRNA conjugate of claim 23wherein the ribozyme RNA of (a)(1) comprises at least 18 nucleotides.27. The polypeptide-engineered mRNA conjugate of claim 23 wherein theribozyme of (a)(1) is a contiguous sequence.
 28. Thepolypeptide-engineered mRNA conjugate of claim 23 wherein the ribozymeRNA of (a)(1) comprises modified BIV-1 TAR RNA and the peptide tag isthe Tat tag peptide or the Tat 2 tag peptide.
 29. Thepolypeptide-engineered mRNA conjugate of claim 23 wherein eachengineered mRNA additionally comprises a coding region for a peptidelinker, wherein the coding region for the peptide linker is locatedbetween (a)(2) and (a)(3).
 30. The polypeptide-engineered mRNA conjugateof claim 29 wherein the region coding for a peptide linker encodes alinker of 30 amino acid residues or more.
 31. The polypeptide-engineeredmRNA conjugate of claim 23 wherein the engineered mRNA further comprisesa ribosome stalling site located between the coding region for a diversepolypeptide and the second PCR primer-binding site.
 32. Thepolypeptide-engineered mRNA conjugate of claim 23 wherein in eachengineered mRNA the ribozyme RNA is the ribozyme of SEQ. ID No.: 5 orSEQ ID NO.
 6. 33. A diverse encoded polypeptide library comprised ofpolypeptide-engineered mRNA conjugates, wherein each encodedpolypeptide-engineered mRNA conjugate in the library comprises:(a) anengineered mRNA which comprises:(1) ribozyme RNA which specificallycovalently links to a peptide in the presence of the remainder of theribozyme; (2) a coding region for the peptide with which the ribozymeRNA of (a) specifically covalently links, wherein the peptide isreferred to as a peptide tag; (3) a coding region which encodes apolypeptide to be expressed; and (4) a second PCR primer-binding site;and (b) the translation product of the engineered mRNA, which comprisesthe polypeptide to be expressed and the peptide tag,wherein the ribozymeRNA of the engineered mRNA is joined to the peptide tag by a covalentbond between the ribozyme RNA of the engineered mRNA and a reactivegroup of the peptide tag.
 34. The diverse encoded polypeptide library ofclaim 33 wherein in each engineered mRNA the ribozyme RNA is theribozyme of SEQ ID NO.: 5 or SEQ ID NO.:
 6. 35. The diverse encodedpolypeptide library of claim 33 wherein the engineered mRNA furthercomprises a coding region for a peptide linker, wherein the codingregion for a peptide linker is located between (a)(2) and (a)(3). 36.The library of claim 33 wherein the ribozyme RNA of (a)(1) is acontiguous sequence.
 37. The library of claim 33 wherein the ribozymeRNA of (a)(1) comprises modified BIV-1 TAR RNA and the peptide tag isthe Tat tag peptide or the Tat 2 tag peptide.
 38. The library of claim33 wherein each engineered mRNA additionally comprises a coding regionfor a peptide linker, wherein the coding region for the peptide linkeris located between (a)(2) and (a)(3).
 39. A method of producing adiverse library of encoded polypeptides, which comprisespolypeptide-engineered mRNA conjugates, comprising the steps of:(a)combining:(1) a library of engineered mRNAs, wherein each engineeredmRNA in the library comprises:(i) ribozyme RNA which specificallycovalently links to a peptide tag in the presence of the remainder ofthe ribozyme sequence; (ii) a coding region for the peptide with whichthe ribozyme RNA of (a)(1)(i) specifically covalently links, wherein thepeptide is referred to as a peptide tag; (iii) a coding region for adiverse polypeptide; and (iv) two PCR primer-binding sites; and (2) anappropriate in vitro translation mixture, thereby producing acombination; (b) maintaining the combination under conditionsappropriate for translation of the engineered mRNA to occur,whereby thetranslation product of the engineered mRNA, which comprises the peptidetag and the diverse polypeptide segment, is produced and joined to theengineered mRNA by a covalent bond between the ribozyme RNA of theengineered mRNA and a reactive group of the peptide tag, therebyproducing polypeptide-engineered mRNA conjugates.
 40. The method ofclaim 39 wherein a region coding for a linker sequence is locatedbetween (a)(1)(ii) and (a)(1)(iii).
 41. The method of claim 39 whereinthe ribozyme RNA of (a)(1)(i) comprises at least 18 nucleotides.
 42. Themethod of claim 39 wherein the ribozyme RNA of (a)(1)(i) is a contiguoussequence.
 43. The method of claim 39 wherein the ribozyme sequencecomprises SEQ ID NO.: 5 or SEQ ID NO.:
 6. 44. The method of claim 39wherein the ribozyme RNA of (a)(1)(i) comprises modified BIV-1 TAR RNAand the peptide tag is the Tat tag peptide or the Tat 2 tag peptide. 45.The method of claim 39 wherein each engineered mRNA additionallycomprises a coding region for a peptide linker, wherein the regioncoding for the peptide linker is located between (a)(1)(ii) and(a)(1)(iii).
 46. A method of identifying members of a diverse encodedpolypeptide library which exhibit a desired activity, wherein membersare polypeptide-engineered mRNA conjugates, comprising the steps of:(a)producing a diverse encoded polypeptide library which comprisespolypeptide-engineered mRNA conjugates by:(i) combining:(1) a library ofengineered mRNAs, wherein each engineered mRNA in the librarycomprises:(a) ribozyme RNA which specifically covalently links to apeptide; (b) a coding region for the peptide with which the RNA of(1)(a) interacts, wherein the peptide is referred to as a peptide tag;(c) a coding region for a diverse polypeptide; and (d) two PCRprimer-binding sites; and (2) an appropriate in vitro translationmixture, thereby producing a combination; (ii) maintaining thecombination under conditions appropriate for translation of theengineered mRNA to occur,whereby the translation product of theengineered mRNA, which comprises the peptide tag and the diversepolypeptide, is produced and joined to the engineered mRNA by a covalentbond between the 5' end of the engineered mRNA and a reactive group ofthe peptide tag, thereby producing polypeptide-engineered mRNAconjugates; (b) enriching the diverse encoded polypeptide library formembers which exhibit a desired activity, thereby producing an enrichedpolypeptide library comprised of polypeptide-engineered mRNA conjugates;(c) amplifying the enriched encoded polypeptide library by:(i) reversetranscribing the engineered mRNA component of the conjugates, therebyproducing the corresponding cDNA; (ii) amplifying and transcribing invitro the corresponding cDNA, thereby producing a pool of amplified,enriched engineered mRNA; (iii) combining the pool of amplified,enriched engineered mRNA with an appropriate in vitro translationmixture, thereby producing a combination; and (iv) maintaining thecombination under conditions appropriate for translation of theengineered mRNA to occur, whereby the translation product of theengineered mRNA is produced and joined to the engineered mRNA by acovalent bond between the ribozyme RNA of the engineered mRNA and areactive group of the peptide tag, thereby producing an amplifiedenriched polypeptide library comprised of polypeptide-engineered mRNAconjugates; (d) repeating steps (b)-(c) as necessary until members whichexhibit the desired activity are present in sufficient number to bedetected; and (e) detecting members which exhibit the desired activity,thereby identifying members which exhibit the desired activity.
 47. Themethod of claim 46 wherein the ribozyme RNA of (a)(i)(1)(a) is onenucleotide.
 48. The method of claim 46 wherein the ribozyme RNA of(a)(i)(1)(a) is from 1 to about 18 nucleotide residues, inclusive,wherein the 1 to 18 nucleotide residues are from the 5' end of theribozyme sequence.
 49. The method of claim 46 wherein the ribozyme RNAof (a)(i)(1)(a) comprises at least 18 nucleotides.
 50. The method ofclaim 46 wherein the ribozyme RNA of (a)(i)(1)(a) is a contiguoussequence.
 51. The method of claim 46 wherein the ribozyme RNA of(a)(i)(1 )(a) comprises modified BIV-1 TAR RNA and the peptide tag isthe Tat tag peptide or the Tat 2 tag peptide.
 52. The method of claim 46wherein the ribozyme RNA is the ribozyme of SEQ ID NO.: 5 or SEQ ID NO.:6.
 53. The method of claim 46 wherein the engineered mRNA furthercomprises a coding region for a linker, wherein the coding for thelinker is located between (a)(i)(1)(b) and (a)(i)(1)(c).
 54. A member ofa diverse encoded polypeptide library which exhibits a desired activity,identified by the method of claim
 46. 55. A polypeptide fragment of apolypeptide-engineered mRNA conjugate, wherein the polypeptide fragmentcomprises a diverse polypeptide and a peptide tag.
 56. An engineeredmRNA fragment of a polypeptide-engineered mRNA conjugate whichcomprisesa) a ribozyme RNA which specifically covalently links to apeptide in the presence of the remainder of the ribozyme; b) a codingregion for the peptide with which the ribozyme RNA covalently links,wherein the peptide is referred to as a peptide tag; c) a coding regionfor a diverse polypeptide; and d) two PCR primer-binding sites.
 57. Anisolated ribozyme that joins an mRNA to the translation product of themRNA.
 58. The isolated ribozyme of claim 57 that comprises:a) SEQ IDNO.: 5; b) SEQ ID NO.: 6; or c) SEQ ID NO.: 7 and SEQ ID NO.:
 8. 59. Theisolated ribozyme of claim 57 wherein the ribozyme comprises anucleotide sequence sufficiently similar to SEQ ID NO.: 5 to result inthe conformation of the ribozyme of SEQ ID NO. 5 or a nucleotidesequence sufficiently similar to SEQ ID NO.: 6 to result in theconformation of the ribozyme of SEQ ID NO.:
 6. 60. A polypeptide-nucleicacid tag conjugate comprising:(a) a polypeptide of interest; (b) apeptide tag linked to the polypeptide of interest, wherein the peptidetag is the substrate of a ribozyme; (c) a ribozyme or a segment of aribozyme for which the peptide tag of (b) is a substrate, wherein theribozyme or ribozyme segment is covalently linked to the peptide tag;and (d) a nucleic acid tag, wherein the nucleic acid tag is linked tothe ribozyme or ribozyme segment.
 61. A method of producing apolypeptide-nucleic acid tag conjugate of claim 60, comprising:(a)combining:(1) a polypeptide of interest which bears a peptide tagwherein the peptide tag is a substrate for ribozyme RNA; (2) ribozymeRNA, wherein the ribozyme RNA specifically covalently links to thepeptide tag of (a)(1) in the presence of the remainder of the ribozymeand has linked thereto a nucleic acid tag; (3) the remainder of theribozyme, thereby producing a combination; and (b) maintaining thecombination under conditions appropriate for ribozyme RNA of (a)(2) toassociate with ribozyme RNA of (a)(3), forming a functional ribozymethat specifically covalently links the peptide tag to ribozyme RNA of(a)(2), whereby a polypeptide-nucleic acid tag conjugate of claim 60 isproduced.
 62. A method of separating a polypeptide from a mixture ofpolypeptides, comprising:(a) tagging the polypeptide at its aminoterminus with a peptide tag, thereby producing a polypeptide-peptidetag, wherein the peptide tag is a substrate for ribozyme RNA, therebyproducing a mixture comprising the polypeptide-peptide tag; (b)combining the mixture produced in (a) with (1) ribozyme RNA which bearsa nucleic acid tag and specifically covalently links to the peptide tagin the presence of the remainder of the ribozyme and (2) the remainderof the ribozyme, under conditions appropriate for ribozyme (b) (1) tospecifically interact with its peptide substrate thereby forming acovalent link with the peptide and tagging the polypeptide with thenucleic acid tag, whereby a mixture comprising a polypeptide-nucleicacid tag conjugate is formed; (c) combining the mixture formed in (b)with a nucleic acid which is a binding partner for the nucleic acid tagof the polypeptide-nucleic acid tag conjugate, whereby the bindingpartner hybridizes with the nucleic acid tag of the conjugate, forming apolypeptide-nucleic acid tag conjugate with the binding partner boundthereto; and (d) separating the polypeptide-nucleic acid tag conjugatewith the binding partner bound thereto from the product of (c), wherebythe polypeptide is separated from the mixture of polypeptides.
 63. Themethod of claim 62 wherein the binding partner is bound to a solidsurface.
 64. A method of detecting a polypeptide of interest in amixture wherein the polypeptide of interest is present in apolypeptide-nucleic acid tag conjugate, comprising:(a) combining amixture to be assessed for the presence of the polypeptide-nucleic acidtag conjugate with a binding partner for the nucleic acid tag, whereinthe binding partner is a nucleic acid sequence sufficientlycomplementary to the nucleic acid tag that the binding partner and thenucleic acid tag of the conjugate bind to one another and remain boundunder the condition used and; (b) detecting whether binding of thenucleic acid tag and the binding partner occurred, wherein if binding isdetected, the polypeptide of interest is detected.
 65. The method ofclaim 64, wherein the binding partner or the nucleic acid tag is labeledwith a detectable moiety.